In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors

Surfactant proteolipid SP-B is a hydrophobic protein of M_r = 8000 identified in organic solvent extracts of pulmonary surfactant. Analysis of the human SP-B RNA predicts that the active surfactant peptide is derived by proteolysis of an M_r = 40 000 precursor. In the present work, characteristics of synthesis, secretion and processing of SP-B were demonstrated in a pulmonary adenocarcinoma cell line by immunoprecipitation of radiolabelled precursors. Treatment of cells with tunicamycin resulted in synthesis and secretion of unglycosylated proSP-B of M_r = 39 000. Immunoprecipitation of protein produced by in vitro translation of human lung poly(A)⁺ RNA detected an M_r = 40 000 protein; the size discrepancy is likely related to cleavage of a leader signal sequence. Endoglycosidase-H-sensitive precursors of M_r = 41 000-43 000, pI = 5.1-5.4 were the first isoforms detected within the cells, and were processed to endoglycosidase-H-resistant isoforms and secreted. Neuraminidase and endoglycosidase-F-sensitive forms of proSP-B were first detected in the media at 60 min as M_r = 42-46 000 isoforms with pI = 4.6-5.1. Proteolytically processed isoforms of proSP-B were detected primarily in the media and were generated by cleavage of an amino-terminal M_r = 16 000 peptide resulting in M_r = 27 000-33 000 isoforms (pH = 5.6-6.8). The M_r = 27 000-33 000 isoforms were sensitive to neuraminidase, resulting in isoforms with pH = 6.0-6.8. Digestion of the M_r = 27 000-33 000 peptide with endoglycosidase-F resulted in isoforms of M_r = 23 000, pH = 6.0-6.8. The endoglycosidase-F-resistant peptide of M_r = 16 000, pI = 4.2-4.4 was identified with an antiserum generated against synthetic peptides derived from the amino-terminal domain, as deduced from the SP-B DNA sequence. Further proteolytic processing of the M_r = 27 000-33 000 isoforms to the M_r = 8000 peptide detected in surfactant was not observed in this cell line. Thus, in the H441-4 cells (a cell line with morphologic features of Clara cells), SP-B is synthesized as a preprotein which undergoes cleavage of a signal sequence and addition of asparagine-linked carbohydrate; proSP-B is secreted by processes which are independent of glycosylation. SP-B peptides of M_r = 27 000-33 000 and M_r = 16 000, representing carboxy and amino-terminal domains, accumulate in the media.